Monoclonal Antibody Drug Development for Alzheimer?s Disease

摘要 阿尔茨海默病(AD)的痴呆是由于大脑中淀粉样蛋白沉积过多引起的 很多年了。淀粉样斑块一旦在大脑中形成，在没有斑块的情况下就是永久性的。 解聚疗法。斑块解聚疗法最有效的形式是单克隆性。 抗AD淀粉样蛋白抗体(MAb)和被动免疫治疗AD是目前研究的热点 在临床试验中进行测试。然而，抗A？单抗不能穿过血脑屏障(BBB)。 因此，必须给予非常高剂量的单抗以降低AD患者的脑内斑块。 转基因小鼠，这些高剂量会导致脑微出血。目前的研究将 从基因上设计一种新形式的抗A？单抗，使其能够通过受体- 介导了血到脑和脑到血两个方向的转运。PI之前已经 通过基因工程获得了一种针对小鼠转铁蛋白受体(TfR)的嵌合单抗，这种单抗可以跨越 通过受体介导的小鼠血脑转运蛋白在血到脑方向的转运 在血脑屏障Fc受体(FCR)上沿脑-血方向穿过血脑屏障。此外，PI还拥有 基因工程，并表达了针对氨基末端的单链抗体(ScFv) A？肽的一部分。本研究将产生一种新的融合蛋白，其中抗A？ 单链抗体与抗小鼠TfR的嵌合单抗的羧基末端融合，这一新的 融合蛋白被命名为TfRMAb-A？scFv双特异性抗体(BSA)。跟随着基因 新的BSA的工程，该蛋白将在COS细胞中瞬时表达，并在 BSA的功能将通过小鼠转铁蛋白受体结合试验和A？结合来证明 化验。然后，BSA将在中国仓鼠卵巢(CHO)细胞中永久表达，随后 通过选择，稀释性克隆，亲和和阳离子交换纯化 层析法。纯化的BSA将用于APPswe/PSEN1(DE9)双转基因的治疗 3个月以上的小鼠。对照组小鼠接受生理盐水或常规高剂量注射。 剂量不通过血脑屏障的抗A？单抗。这些小鼠将接受神经行为评估， 普鲁士蓝检测血浆A？水平、脑A？斑块含量和脑内微量出血 组织化学。这项研究将提供必要的临床前药理学来支持IND 申请使用基因工程融合抗体治疗阿尔茨海默病 通过受体介导跨越血脑屏障，结合和解聚AD的A？淀粉样斑块。与公共卫生的相关性 阿尔茨海默病(AD)的痴呆是由Aβ淀粉样蛋白在脑组织中沉积引起的 大脑中的淀粉样斑块一旦在大脑中形成，它就是永久性的。 斑块解聚治疗的缺失。最有效的斑块形式 去聚集疗法是一种抗Aβ淀粉样肽的单抗 AD的被动免疫治疗目前正在进行临床试验； 然而，抗A？单抗不能穿过血脑屏障(BBB)。现在 研究将在AD转基因小鼠身上测试一种新的基因工程的有效性 融合抗体既能通过受体介导穿过血脑屏障，又能结合和 解聚AD的淀粉样斑块。

ABSTRACT The dementia of Alzheimer's disease (AD) is caused by the deposition of A¿ amyloid in brain over many years. Once the amyloid plaque forms in brain, it is permanent in the absence of plaque disaggregation therapy. The most potent form of plaque diasaggregation therapy is a monoclonal antibody (MAb) against the A¿ amyloid peptide of AD, and passive immune therapy of AD is currently being tested in clinical trials. However, the anti- A¿ MAb does not cross the blood-brain barrier (BBB). Consequently, very high doses of MAb must be administered to lower plaque in brain of AD transgenic mice, and these high doses causes brain microhemorrhage. The present research will genetically engineer a new form of anti-A¿ MAb that is enabled to cross the BBB via receptor- mediated transport in both the blood-to-brain and brain-to-blood directions. The PI has previously genetically engineered a chimeric MAb against the mouse transferrin receptor (TfR) that crosses the BBB in the blood-to-brain direction via receptor-mediated transport on the mouse BBB TfR, and also crosses the BBB in the brain-to-blood direction on the BBB Fc receptor (FcR). In addition, the PI has genetically engineered, and expressed a single chain Fv (ScFv) antibody against the amino terminal portion of the A¿ peptide. The present research will produce a new fusion protein, wherein the anti-A¿ ScFv is fused to the carboxyl terminus of the chimeric MAb against the mouse TfR, and this new fusion protein is designated the TfRMAb-A¿ScFv bi-specific antibody (BSA). Following the genetic engineering of the new BSA, the protein will be transiently expressed in COS cells, and the bi- functionality of the BSA will be demonstrated with a mouse TfR binding assay and an A¿ binding assay. The BSA will then be permanently expressed in Chinese hamster ovary (CHO) cells, followed by selection, and dilutional cloning, and purification with affinity and cation exchange chromatography. The purified BSA will then be used to treat APPswe/PSEN1(dE9) double transgenic mice over 3 month period. Control mice will be treated with either saline or with the conventional high dose anti-A¿ MAb that does not cross the BBB. The mice will be evaluated for neurobehavior, plasma A¿ levels, brain A¿ plaque content, and brain micro-hemorrhage using Prussian blue histochemistry. This research will provide the necessary pre-clinical pharmacology to support an IND filing for the treatment of humans with AD using genetically engineered fusion antibodies that both cross the BBB via receptor-mediation and bind and disaggregate A¿ amyloid plaque of AD. RELEVANCE TO PUBLIC HEALTH The dementia of Alzheimer's disease (AD) is caused by the deposition of A¿ amyloid in brain over many years, and once the amyloid plaque forms in brain, it is permanent in the absence of plaque disaggregation therapy. The most potent form of plaque diasaggregation therapy is a monoclonal antibody (MAb) against the A¿ amyloid peptide of AD, and passive immune therapy of AD is currently being tested in clinical trials; however, the anti- A¿ MAb does not cross the blood-brain barrier (BBB). The present research will test in AD transgenic mice the efficacy of a new genetically engineered fusion antibody that both crosses the BBB via receptor-mediation and binds and disaggregates A¿ amyloid plaque of AD.